The H295R in vitro cell line produces the majority of the steroidogenesis, for which reason it is commonly used as a screening tool for endocrine disrupting chemicals. Simultaneous determination of the precursor cholesterol and key steroid hormones could give a broad insight into the mechanistic disruption of the steroidogenesis. Steroid hormones have primarily been extracted from H295R incubation medium by means of liquid-liquid extraction (LLE) and the obtained recoveries and matrix effects have typically not been stated or assessed. In the present study a solid-phase extraction (SPE) method was developed and validated for the simultaneous extraction of cholesterol and five key steroid hormones pregnenolone, 17-hydroxyprogesterone, testosterone, cortisol and aldosterone from H295R incubation medium, and finally detected by LC-MS/MS. Cholesterol was recovered at a level of 55.7%, while steroid hormone recoveries ranged from 98.2 to 109.4%. Matrix effects varied between -0.6% and 62.8%. Intra-day precision was deemed acceptable, but the inter-day precision for pregnenolone and aldosterone exceeded the precision limit of 15% RSD. Although LLE has been the most frequently used extraction method in H295R studies, however, our investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE.

A multi-residue pressurised liquid extraction (PLE) methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in solid environmental matrices. The PLE methodology is combined with solid phase extraction as clean-up using liquid chromatography coupled to tandem mass spectrometry applying electrospray ionisation for detection. The samples were freeze-dried prior to extraction. The absolute recoveries for soil and sediment ranged from 71 to 123% (relative standard deviation (RSDs) below 16%) and in the range 94–133% (RSDs 9–35%) for poultry manure. The final method allowed for the detection of four ionophores down to a few hundred ng kg−1 in natural solid matrices with limit of quantifications (LOQs) being 0.96, 0.87, 0.98, and 0.64 μg kg−1 in soil for lasalocid, monensin, salinomycin, and narasin, respectively. Corresponding LOQs in sediment were 1.28, 1.34, 1.39, and 0.78 μg kg−1 for the respective ionophores, while in manure the LOQs were 0.98, 1.01, 1.45, and 1.01 μg kg−1.

Dehydration process is an important histotechnical method of tissue samples undergoing histopathological analysis. Using different alcohols and intermedium, the samples are processed for diffusing the water out of the tissue. Impregnation with paraffin of the tissue in the later stagesis crucial for tissue stability in the remaining histotechnical methods. Sakura Tissue-Tek VIP 6 is atraditional dehydration instrument that realizes dehydrations during longer times with xylene asthe intermedium. However, LOGOS is another dehydration instrument operating with shorter times using Histolab Clear, a more environmental and cheaper intermedium.In the current study tissue samples were used from three different patient cases of each tissue:cutis, breast, intestine and prostate. Two different, new LOGOS- dehydration programs with Histolab Clear as intermedium were tested and validated in accordance with specific qualitycriteria. Parallel were tissue samples dehydrated from the same tissues in the traditional routineprogram, in order to evaluate by comparison, sections and staining quality of the new programs.Immunohistochemistry and routine staining were used for evaluation. The pathologist approved allroutine stains and disapproved some immunohistochemical analyses, depending on poorer staining and the quality of the sections.Because the amounts of the samples were insufficient in some cases, the number of the samples were determined to be expanded in order to investigate if the error occurs systematically or onlyto individual cases. Insufficient dehydration seemed to be fundamental as a cause of inferior results. Therefore, even the time of dehydration in Histolab Clear will increase by one hour, in the further validation. Optimization of dehydration time is required for approval of immunohistochemical analyzes. These analyzes are very important and definitive for the treatment of the patient, and therefore the new dehydration programs must give at least as good results as the conventional.

Transplantation is based on a satisfactory matching of the patient and donor genes for Human Leukocyte Antigen (HLA), which increases the chance of a successful transplantation. HLA gives individual cell surface markers. The Major Histocompatibility Complex (MHC) region, encoding HLA in humans, is the most polymorphic in the human genome. The genes are located on chromosome six and consists of 200 genes. Those genes encode protein products essential for the acquired immune system. MHC molecule’s role is to represent foreign substance for B- and T-lymphocytes. MHC is an important system as it contributes to the activation of the immune system to combat viruses, bacteria, parasites and cancer cells. HLA-typing is determined through certain antigens in the HLA system. The classical transplantation antigens are HLA-A, -B, -C, -DR, -DQ and -DP. By amplifying the DNA with sequence specific primers in the Polymerase Chain Reaction (PCR), the amplicons can be detected and alleles present in the patient genome can be determined. The purpose of this study was to compare occurrence of non-specific DNA binding using master mix where Ampli Taq DNA polymerase is added and master mix with polymerase included in the PCR. Samples from 16 patients were tested with both master mix- solutions. The analyses were performed with primer plates for HLA-A, HLA-B and HLA-DRP1. The results showed that the master mix with Taq polymerase included should be applied, because it gave clearer specific band, better image quality and gave weaker and approximately 30% fewer non- specific DNA binding compared to the master mix with added Taq polymerase.

Type 1 diabetes (T1D) is regarded as an autoimmune disease. Beta cells, which produces insulin in pancreas are attacked by islet cell antibodies (ICA). This leads to gradual destruction of the beta cell, which in turn cause high level of glucose in the blood because the regulator "insulin" has disappeared. In that case the patient needs to be treated lifelong with insulin. It has been shown that the ICA reactivity consisting of reactivities against different autoantigens such as: insulin autoantigen (IAA), glutamic acid autoantigen (GAD), insulinoma antigen-2 autoantigen (IA-2) and most likely also zinc transporter autoantigen (ZnT8). Determination of ICA in serum samples is important for the classification of diabetes, prediction of T1D and the development of autoimmune therapies.

Nowadays screening of ICA is performed with ”Medipan ICA screen” which is a commercial enzyme- linked immunosorbent assay (ELISA). Positives samples are further analysed by ELISA with the indirect immunofluorescence method (IF) to ensure a final positive answer.

The purpose of this study was to evaluate and compare a new commercial ELISA kit ”RSR 2screen” with the Medipan ICA screen for use it in routine analysis to evalute if it has the same / higher specificity and sensitivity, and lower price compared with Medipan ICA screen.

Serum samples from a control group (n = 199) and a patient’s group diagnosed with T1D (n = 100 were analyzed with both ELISA methods. The results were statistically evaluated to set a threshold value for positivity and to evaluate the method's sensitivity and specificity. The result showed that both ELISA- methods gave the same sensitivity (93%) and specificity (97.5%) and a high concordance (98.7%) was achieved. Analytical price per sample for the RSR 2screen was 4.2% lower than for the Medipan ICA screen. RSR 2screen can be used instead of Medipan ICA screen.

Venous thromboembolism that cause blood clotting in blood vessels, prevent blood circulation, depending on changes in one or more of the coagulation factors II, VII, IX and X. Patients who have had a blood clot or cardiovascular diseases are treated with oral anti-vitamin K (Warfarin®) to reducing and prevent relapse. Warfarin is also used as a preventive treatment before the disease. An overdose of Warfarin® may cause bleeding-complications and low dose cause blood clotting. The dosage of the drug is controlled by measuring prothrombin in plasma. The aim of this study was to investigate if prothrombin-complex value changes due to re-spinning and re-analysis after six hours. Fitty whole blood samples from warfarin-treated patients were divided into three subgroups, those with protrombinkomplex-values of 2-4 (n=20), >4 (n=15) and <2 (n=15). The samples were centrifugated and measured (Method A), re-centrifugated and measured (Method B) or re-analysed after six hours (Method C). All results were compared in a Bland-Altman plot as follows: Method B vs. Method A and Method C vs. Method A. The scatter graph yielded a strong correlation between Method A and Method B (R2=0.9984) and Method A and Methods C (R2=0.9977). The results from t-test showed a significance level (p<0.001) for both analyses (statistical significance=p<0.05). In this study we showed that prothrombin complex value ware stable after re-centrifugation and re-measurement after six hours. Statistical calculations yielded a strong correlation between the methods (A, B, C), and there was no significance difference between the methods.

INTRODUCTION: Haemophilia B is caused by a heterogeneous spectrum of mutations. Mutation characterization is important in genetic counselling, prenatal diagnosis and to predict risk of inhibitor development.

AIMS: To study the mutation spectrum, frequency of unique recurrent mutations, genotype-phenotype association and inhibitor development in a population-based study of the complete Swedish haemophilia B population.

METHODS: The study included, facilitated by centralized DNA diagnostics, the complete registered Swedish haemophilia B population (113 families: 47 severe, 22 moderate and 44 mild), each represented by a single patient. Mutation characterization was performed by conventional sequencing of all exons and haplotyping by genotyping of single nucleotide variants and microsatellites.

RESULTS: A mutation was found in every family: eight had large deletions, three had small deletions (<10 base pair) and 102 had single base pair substitutions (69 missense, 26 nonsense, four splice site and three promoter). Ten novel mutations were found and were predicted to be deleterious. Sixteen mutations (one total gene deletion, 14 substitutions and one acceptor splice site) were present in more than one family. Of the single nucleotide mutations (37/102), 36% arose at CpG sites. Haplotyping of families with identical mutations and present analyses showed that the frequency of unique mutations was at least 65%. Inhibitors developed in 9/47 (19%) patients with severe haemophilia B.

CONCLUSION: The spectrum of haemophilia B mutations reveals at least 65% of the families carry a unique mutation, but with more inhibitor patients than reported internationally, probably as a result of many 'null' mutations.

Heart failure is a term for a group of complex symtoms characterized by reduced heart function. One of these syndromes, referred to as heart failure with preserved ejection fraction (HFpEF), has increased in prevalence compared to other types of heart failures during the recent years. A concern is the difficulty in diagnosing patients with HFpEF, since current tools are considered insufficient. The aim of this thesis was to examine Peak Oxygen Uptake (VO2peak) in relation to Total Heart Volume (THV) among heart failure patients with preserved ejection fraction (HFpEF, EF >40 %) compared to healthy controls. THV was acquired by delineating images acquired using cardiovascular magnetic resonance imaging, while VO2peak was measured in oxygen curves acquired from cardiopulmonary exercise tests. Ratios were calculated by dividing VO2peak with THV. In order to determine if blood hemoglobin concentration (b-Hb) could affect the ratio, ratios were adjusted to b-Hb using an adjusting factor. Mean THV was nearly 250 ml larger in HFpEF patients compared to the controls. Patients’ mean VO2peak was more than 1000 ml lower compared to the controls. Mean VO2peak/THV ratio calculated for the patients were less than half of that calculated for the controls. Adjusting the ratio to b-Hb did not affect the ratios significantly. The study was limited by the size of the test group, but the findings suggest that a VO2peak/THV ratio can be used to separate HFpEF patients from healthy controls.

Aim: To examine the existing evidence in identifying risk indicators in the etiology of peri-implantitis. Material and methods: A literature search was performed in MEDLINE via PubMed database of the US National Library of Medicine, for articles published until October 2014 using Medical Subject Heading search terms + free text terms and in different combinations. Results: The microbiota associated with peri-implantitis is complex, demonstrating differences and similarities to the one seen at periodontitis sites. Plaque accumulation at dental implants triggers the inflammatory response leading to peri-implant mucositis/peri-implantitis. Individuals with a history of periodontal disease and smokers have an increased risk of developing peri-implantitis. There is some evidence to support the role of genetic polymorphism, diabetes, and excess cement as risk indicators for the development of peri-implantitis. There is also evidence to support that individuals on regular maintenance are less likely to develop peri-implantitis and that successful treatment of periodontitis prior to implant placement lowers the risk of peri-implantitis. Conclusions: Plaque accumulation at implants will result in the development of an inflammation at implants. A history of periodontal disease, smoking, excess cement, and lack of supportive therapy should be considered as risk indicators for the development of peri-implantitis.

Background: To prevent bacteria from forming biofilm on medical devices is an important challenge.Biofilm formation is associated with decreased susceptibility to antibioticsand the development of antibiotic resistance. The problem has to be solved by both preventing the formation of biofilm and development of new antibiotics directed to the formation of biofilm. Antibacterial coating on medical devices is a preventive action to decrease the risk of catheter related infections. The Bactiguard coating is one example, and has been clinically proven to reduce infections. But if the Bactiguard coating affects formation of microbal biofilm or production of extracellular polymeric substances (EPS) is not yet fully understood.

Methods: The amount of biofilm formed on coated and uncoated surface was compared by staining of EPS. Three different bacterial species were cultured separately and together.

Results: All three species of bacteria did form biofilm on Bactiguard coated surface. However, there was significantly less bacterial growth on the Bactiguard coated microtiter plates compared to the uncoated. Quantification of EPS was not successful. Colonization of S. epidermidis appeared differently on Bactiguard coated microscope slides after 4-8 h of culture.

Discussion: The antibacterial effects of Bactiguard coated catheters seem to be more connected to inhibited adherence of bacteria to the surface. The coating has to be optimized for further study on microtiterplates.

This paper presents the development and evaluation of a methodology for extraction, clean-up and analysis of three key corticosteroids (aldosterone, cortisol and corticosterone) in polar bear hair. Such a methodology can be used to monitor stress biomarkers in polar bears and may provide as a useful tool for long-term and retrospective information. We developed a combined pressurized liquid extraction (PLE)-solid phase extraction (SPE) procedure for corticosteroid extraction and clean-up followed by high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis. This procedure allows for the simultaneous determination of multiple steroids, which is in contrast to previous polar bear studies based on ELISA techniques. Absolute method recoveries were 81%, 75% and 60% for cortisol, corticosterone and aldosterone, respectively. We applied the developed method on a hair sample pooled from four East Greenland polar bears. Herein cortisol and corticosterone were successfully determined in levels of 0.32 +/- 0.02 ng/g hair and 0.13 +/- 0.02 ng/g hair, respectively. Aldosterone was below limit of detection (LOD<0.17 ng/g). The cortisol hair concentration found in these East Greenland polar bears was consistent with cortisol levels previously determined in the Southern Hudson Bay and James Bay in Canada using ELISA kits. (C) 2016 Elsevier B.V. All rights reserved.